Product and method for combating swine dysentery

ABSTRACT

A substituted pyrazine having the formula ##STR1## is useful for treating and preventing swine dysentery. It may be administered to swine in an effective but non-toxic amount in the form of the drug per se, or a feed composition. Such feeds may be made with the aid of premixes containing the said compound.

DESCRIPTION

This invention relates to the treatment and prevention of swine dysentery, and to products for use therein.

Swine dysentery (also known as vibrionic dysentery, bloody scours, or hemorrhagic dysentery) is an enteric disease primarily characterised by muco-hemorrhagig diarrhea with lesions usually restricted to the large intestine. The disease is worldwide and is a major disease problem among swine producers all over the world.

The earlier consensus was the Vibrio coli was the primary causative agent. Recent evidence suggests, however, that a spirochete, Treponema hyodysenteriae is involved with the disease and may in fact be the primary etiologic agent.

Currently, control measures are based on constant feeding of antibacterial agents with therapy based on use of high levels of these drugs. The drugs used include furazolidone, neomycin, oxytetracycline, tylosin, carbadox, virginiamycin and arsanilic acid. Unfortunately, these drugs give erratic results, even when used at abnormally high levels.

Accordingly there is a continuing need for new drugs of low toxicity and high potency to combat swine dysentery.

It has now been discovered that a substituted pyrazine of that formula: ##STR2## hereinafter referred to as "D-compound" is useful in veterinary therapy for the treatment and prophylaxis of swine dysentery. This compound selectively combats the swine dysentery-causing organisms without deleteriously affecting the balance of other desired organisms, e.g. in the internal biological system of swine, such as the intestinal flora.

The antibiotic substance D-aspergillic acid which has been isolated from cultures of Aspergillus flavus conform to the aforesaid formula.

In the method of the present invention, the D-Compound is administered to swine in an amount effective to combat dysentery. It can be advantageously incorporated in a swine feed-stock to provide a swine feed composition for combatting dysentery. It can be incorporated in the swine feed-stock generally at a level of from about 25 g/ton to about 500 g/ton. The preferred level, however, particularly in the absence of the disease, is about 100 to 200 g/ton for prophylaxis, advantageously for a period of 3 to 21 days. However, it there has been an outbreak of the disease, or if new animals whose history is not known have been introduced into a herd, the higher level of 200 to 500 g/ton is preferred until the health of the herd is assured. Generally, however, the prophylactic treatment is continued until the animals are ready for market. The D-Compound can also be administered by incorporation into drinking water provided for swine.

The D-Compound is useful for combatting swine dysentery-causing organisms, e.g. dysentery caused by Vibrio or Treponema organisms, or both. The D-Compound is of a low order of toxicity and is suitable for use by oral administration for prophylactic or therapeutic treatment of swine dysentery. It is not nitrogenic.

A swine feed-stock for oral administration of D-Compound according to this invention can be readily prepared by intimately admixing the D-Compound alone or as a premix with a conventional swine feed composition to provide a homogeneous feed product.

The term feed-stock means any food provided for the swine. Preferably the D-Compound is thoroughly mixed with the feed-stock so that it is uniformly dispersed throughout. However, it may also be sprinkled on the daily food supplies in the form of a powder or as pellets. Thus, the invention is not limited to any particular mode of administration.

The following Example illustrates the invention.

EXAMPLE

The D-Compound was tested against five strains of Vibrio cholerae at concentrations of 10, 30 and 100 micrograms per milliliter. The results are given in Table 1 below.

Tests were also run to see if the compound was effective against Vibrio cholerae El Tor Ogawa 6 in the presence of sewage. Sewage samples were obtained from the sewer system of the city of Modena, Italy. They were centrifuged to separate solids and the supernatant liquid was used in the tests. The results are given in Table 2.

At 10 mcg/ml of D, there was no growth of 3 of the organisms after 48 hours, and only marginal growth of the remaining two at 100 mcg/ml.

D was tested in vitro against Treponema hyodysenteriae by a known method. The minimum inhibitory concentration (the lowest concentration of compound in a dilution series where growth is inhibited) was 0.1 mcg/ml. The minimum bactericidal concentration (the lowest concentration of compound in which no viable treponemes are observed upon dilution and subculture from the broth onto blood agar plates) was greater than 0.1 mcg/ml but less than 1 mcg/ml.

The compound was tested for acute toxicity by several modes of administration in four species, namely mice, rat, guinea pig, and rabbit. The compound was found to be of a low order of toxicity. The results are given below in Tables 3, 4, 5 and 6.

In view of the favourable acute toxicity data, the compound was administered orally in sub-acute, but relatively large, doses to mice and rats for 15 days. Data were collected on the effects on death rate, weight, liver, and kidneys. The data are given in Tables 7 and 8.

In view of the favourable results on chronic toxicity, a teratogenic study was conducted with male and female mice and rats. The number of young delivered live at birth was comparable with controls. No malformations in either group were observed. The data are given in Table 10.

                  TABLE 1                                                          ______________________________________                                         Con-       Effect on Various Strains of Vibrio Cholerae                              centra-  Classical                                                                               Classical                                                                             El Tor                                                                               El Tor                                                                               El Tor                              Com-  tion     Inaba    Ogawa  Ogawa Ogawa Inaba                               pound μg/ml 35       41     6     8     4                                   ______________________________________                                         D     100      -        -      ±  -     ±                                      30       -        -      -     -     ++                                        10       -        -      ++    -     ++                                  ______________________________________                                          -  No growth after 48 hours at 37° C.                                   ±  Just noticeable growth                                                   +  Evident growth but to a smaller extent than in untreated control            experiments                                                                    ++  Same degree of growth as in untreated control experiments.           

                  TABLE 2                                                          ______________________________________                                                   Concentration                                                                           Effect After                                                Sample      of D       24 hours 48 hours                                                                              5 days                                  ______________________________________                                         Control + Vibrion                                                                          --         +++      +++    +++                                     Sewage      --         ---      ---    ---                                     Sewage + Vibrion                                                                           --         +++      +++    +++                                     Sewage + Vibrion                                                                            5γ/ml                                                                              ---      ---    ---                                     Sewage + Vibrion                                                                           10γ/ml                                                                              ---      ---    ---                                     Sewage + Vibrion                                                                           20γ/ml                                                                              ---      ---    ---                                     Sewage + Vibrion                                                                           30γ/ml                                                                              ---      ---    ---                                     ______________________________________                                    

                  TABLE 3                                                          ______________________________________                                          Acute Toxicity of D in female Mice                                            Dosage   Dead/Treated Animals after                                            mg/kg    1 day     2 days    4 days  7 days                                    ______________________________________                                                Endoperitoneal Administration                                           2000     6/6                         6/6                                       1000               6/6               6/6                                        500                                 6/12                                       250                                 0/18                                             Esophageal Administration                                               0 (x)                              0/6                                         4000                     1/12      1/12                                        2000                               0/12                                        1000                               0/12                                        ______________________________________                                          (x) By gastric lavage and receiving only the vehicle.                    

                                      TABLE 4                                      __________________________________________________________________________      Acute Toxicity of D in the Rat                                                __________________________________________________________________________     a.                                                                               First Experiment                                                                Route of    Dead/Treated                                                                           Body weight                                                                           (m ± SEM)                                                                          Statistical                               Sex                                                                               Administration                                                                         mg/kg                                                                              within 21 days                                                                         in g. start                                                                           Termination                                                                           Significance(.sup..)                      __________________________________________________________________________     M  Esophageal                                                                             4000                                                                               0/4     234.5 ± 13.8                                                                       288.7 ± 13.8                                                                       t 0.05                                    M  Esophageal                                                                             0 (x)                                                                              1/4     233.7 ± 3.7                                                                        331.0 ± 0.5                                   F  Esophageal                                                                             4000                                                                               0/4     201.2 ± 4.2                                                                        238.2 ± 12.1                                                                       t 0.05                                    F  Esophageal                                                                             0 (x)                                                                              1/4     189.2 ± 3.9                                                                        230.0 ± 10.5                                  M  Endoperitoneal                                                                          500                                                                               1/4     234.0 ± 6.2                                                                        314.3 ± 10.3                                                                       t 0.05                                    M  Endoperitoneal                                                                         0 (x)                                                                              0/4     230.0 ± 5.7                                                                        324.0 ± 8.7                                   F  Endoperitoneal                                                                          500                                                                               2/4     206.2 ± 8.7                                                                        286.0 - 272.0                                                                         t 0.05                                    F  Endoperitoneal                                                                         0 (x)                                                                              0/4     207.5 ± 4.3                                                                        253.5 ± 7.7                                   __________________________________________________________________________      (x) Only the vehicle was administered by the same route.                       (.sup..) Student's t test.                                                    b.                                                                               Second Experiment                                                                 Route of       Dead/Treated                                                                            Body weight                                                                            (±SE)                                  Sex  Administration                                                                          mg/kg within 7 days                                                                           in g. start                                                                            Termination                               __________________________________________________________________________     M    Esophageal                                                                              4000  0/4      222.5 ± 6.2                                                                         231.7 ± 15.7                           F    Esophageal                                                                              4000  0/4      252.0 ± 16.6                                                                        253.5 ± 12.1                           M    Intraperitoneal                                                                          500  2/4      226.2 ± 6.8                                                                         225.0 - 212                               F    Intraperitoneal                                                                          500  0/4      232.5 ± 5.9                                                                         218.2 ± 7.0                            __________________________________________________________________________     c.                                                                               Cumulative Data Regardless of Animal Sex                                     Route of                                                                       Administration                                                                               mg/kg Dead/Treated within 7 days                                 __________________________________________________________________________     Esophageal    0 (x) 0/8                                                        Esophageal    4000  0/16                                                       Intraperitoneal                                                                              0 (x) 0/8                                                        Intraperitoneal                                                                               500  4/16                                                       __________________________________________________________________________      (x) Only the vehicle was administered.                                   

                  TABLE 5                                                          ______________________________________                                         Acute Toxicity of D in the Guinea Pig                                          By Esophageal Administration                                                   Dosage                                                                         mg/kg        Dead/Treated within 21 days                                       ______________________________________                                          500         0/4                                                               1000         1/4                                                               2000         5/6                                                               4000         6/6                                                               0(x)          0/13                                                             ______________________________________                                          (x) Only the vehicle was administered.                                   

                  TABLE 6                                                          ______________________________________                                         Acute Toxicity of D in the Rabbit                                              By Esophageal Administration                                                   Dosage  Dead/Treated Body Weight (m ± SE)                                   mg/kg   within 7 days                                                                               in g. start Termination                                   ______________________________________                                         2000      0/2.sup.(.)                                                                               2250 - 2150 2180 - 2140                                   1000    0/4          2037 ± 104.3                                                                            1922.5 ± 71.5                              0(x)    0/4          2135 ± 75                                                                               2262 ± 215                                  500    0/2          2000 - 2100 1650 - 1550                                   ______________________________________                                          (x) Only the vehicle was administered.                                         (.sup..) There were two dead out of seven treated animals, within 4 days.

                  TABLE 7                                                          ______________________________________                                         Subacute Toxicity of D in the Mouse                                            Daily Dose: 500 mg. CO-1 by gastric lavage for 15 days                         ______________________________________                                                        % Body                                                                         Weight     Fresh Organ-to-Body                                          Dead/  Change     Weight Ratio                                         Oral Treatment                                                                           Treated  (m + SE)   Liver   Kidneys                                  ______________________________________                                         Vehicle   0/10     20.4 ± 4.2                                                                             5.2 ± 0.2                                                                           1.4 ± 0.1                             CO-1,     0/10     -8.1 ± 3.9                                                                             5.9 ± 0.3                                                                           1.5 ± 0.1                             500 mg/kg/day                                                                  ______________________________________                                         a. Mortality and Body Weight                                                   Daily Dose: 1 g/kg/day for 15 days                                                                 Dead/    % Body                                            Oral Treatment      Treated  Weight Change                                     ______________________________________                                         Vehicle (H.sub.2 O) 0/12     24.54 ± 0.64                                   CO-1 in H.sub.2 O, 1 g/kg/day                                                                      2/12     18.5 ± 0.75                                    Vehicle (adraganth gum) (x)                                                                        0/12     25.04 ± 1.18                                   CO-1 in adraganth gum                                                                              3/12     16.27 ± 1.31                                   ______________________________________                                         b. SGOT and SGPT (24 hrs. after last dose)                                                                  Units/ml                                          Oral Treatment      SGOT     SGPT                                              ______________________________________                                         Vehicle:                                                                       Water               116      4                                                 Adraganth gum       119      6                                                 CO-1 in water       124      9                                                 CO-1 in adraganth gum                                                                              132      10                                                ______________________________________                                    

                  TABLE 8                                                          ______________________________________                                         Subacute Toxicity of D in Female Rats                                          ______________________________________                                         Daily Dose: 22 g/kg/day of D by gastric lavage for 21 days                                      Body Weight in g (m ± SE)                                  Oral Treatment                                                                            Dead/Treated                                                                               Start      Termination                                  ______________________________________                                         Vehicle    2/6(x)      200.0 ± 4.1                                                                            233.2 .sub.5/8 5.1                           D, 2 g/kg/day                                                                             1/6(x)      204.1 ± 2.0                                                                            210.6 ± 9.6                               ______________________________________                                          (x) Death caused by a mistake in esophagus incannalutation. This diagnosi      was confirmed at the postmortem examination.                             

    Daily Dose: 2 g/kg/day of D by gastric lavage for 21 days                      Oral    Average Percent Weight of Fresh Organs (m + SE)                        Treatment                                                                              Lung        Liver        Kidneys                                       ______________________________________                                         Vehicle 0.85 ± 0.06                                                                             3.45 ± 0.07                                                                              0.95 ± 0.04                                (3 animals)                                                                    D,      1.07 ± 0.09NS                                                                           4.54 ± 0.10NS(x)                                                                         1.04 ± 0.03NS                              (5 animals)                                                                    ______________________________________                                          (x) Death caused by a mistake in esophagus incannalutation. This diagnosi      was confirmed at the post mortem examination.                            

In view of the favourable sub-acute toxicity, the chronic toxicity in female mice was studied. The results are given in Table 9.

                  TABLE 9                                                          ______________________________________                                         Chronic Toxicity in the Female Mouse                                           Daily treatment by gastric lavage for                                          18 weeks (4.5 months)                                                          ______________________________________                                         a. Mortality and Body Weight                                                              Dead/   Body Weight in g(m ± SE)                                 Oral Treatment                                                                              Treated   Start      Termination                                  ______________________________________                                         Vehicle      3/10      28.2 ± 1                                                                               33.0 ± 1.1                                D, 500 mg/kg/day                                                                            2/10      30.4 ± 0.9                                                                             30.0 ± 0.7                                D, 250 mg/kg/day                                                                            0/10      27.3 ± 0.5                                                                             26.7 ± 0.7                                ______________________________________                                         b. Urine excretion.                                                            Urine amount excreted by 6 animals in 6 hours                                  Oral Treatment  Urine Amount (ml)                                              ______________________________________                                         Controls        6                                                              D, 500 mg/kg/day                                                                               7                                                              D, 250 mg/kg/day                                                                               6.5                                                            ______________________________________                                         c. Blood glucose. Mean values for 6 animals. Blood                             samples were taken 24 hours after the last dose                                Oral treatment   Blood Glucose                                                 ______________________________________                                         Controls         1.14                                                          D, 500 mg/kg/day 1.06                                                          D, 250 mg/kg/day 1.10                                                          ______________________________________                                         d. SGPT and SGOT. Mean values for 6 animals. Blood samples                     were taken 24 hours after the last dose                                                         Units/ml                                                      Oral Treatment     SGOT    SGPT                                                ______________________________________                                         Controls           125     5                                                   D, 500 mg/kg/day   159     6                                                   D, 250 mg/kg/day   118     5                                                   ______________________________________                                         Chronic Toxicity of D in the Female Mouse                                      e. Fresh Weights of Organs                                                     Oral  Fresh Organ-to-Body-Weight Ratio                                         Treat-                                                                               (m ± SE, 4 animals)                                                   ment  Kidneys    Heart      Liver   Lungs                                      ______________________________________                                         Con-  0.938 ± 0.044                                                                          0.481 ± 0.055                                                                          4.57 ± 0.15                                                                         0.674 ± 0.044                           trols                                                                          D, 500                                                                         mg/   1.07 ± 0.04                                                                            0.47 ± 0.02                                                                            4.66 ± 0.91                                                                         1.011 ± 0.110                           kg/day                                                                         D, 250                                                                         mg/   0.87 ± 0.08                                                                            0.60 ± 0.08                                                                            4.57 ± 0.25                                                                         0.731 ± 0.035                           kg/day                                                                         ______________________________________                                    

                                      TABLE 10                                     __________________________________________________________________________      Teratogenetic study                                                           __________________________________________________________________________     a. Animal Species: Mouse, Male and female mice housed together for 10          days.                                                                          Oral treatment from 3rd day to 13th days.                                                Pregnant/                                                                             No. of living                                                                            Body Weight                                                                           No. of                                                 Treated                                                                              Foetuses per                                                                             of Foetuses                                                                            Foetuses with                                Oral treatment                                                                           Animals                                                                              Delivery (m ± SE)                                                                     in g (m ± SE)                                                                       malformations                                __________________________________________________________________________     D, 250 mg/kg/day                                                                         3/10(x)                                                                              10.3 ± 0.6                                                                            1.42 ± 0.05                                                                         0                                            Controls  9/10   9.0 ± 0.9                                                                            1.46 ± 0.07                                                                         0                                            __________________________________________________________________________      (x) On the basis of our wide experience, the above result might be casual      The study should be repeated to determine whether CO1 actually prevents        pregnancy.                                                               

    b. Animal Species. Rat. Same experimental conditions as with the mouse.                  Pregnant/                                                                            No. of Living                                                                            Body Weight                                                                            No. of                                                 Treated                                                                              Foetuses per                                                                             of Foetuses                                                                            Foetuses with                                Oral Treatment                                                                           Animals                                                                              Delivery (m ± SE)                                                                     in g (m ± SE)                                                                       malformations                                __________________________________________________________________________     D, 250 mg/kg/day                                                                         7/10  10.8 ± 0.86                                                                           7.08 ± 0.19                                                                         0                                            Controls  6/10  11.3 ± 1.12                                                                           6.82 ± 0.40                                                                         0                                            __________________________________________________________________________ 

I claim:
 1. A method for the treatment or prophylaxis of swine dysentery which comprises administering to swine an effective non-toxic amount of ##STR3##
 2. A swine feed composition having activity against swine dysentery-causing organisms comprising an edible swine feed carrier and dispersed therein, an effective non-toxic amount of ##STR4##
 3. A feed composition according to claim 2 in which the pyrazine is present in an amount from about 25 g to about 500 g per ton of whole swine feed.
 4. A comestible for ad libitum feeding to swine comprising a comestible swine feed carrier and dispersed therein, from about 100 to 200 g per ton of whole swine feed, ##STR5##
 5. A comestible according to claim 4 in the form of a beverage. 